Low-Density Lipoprotein Receptor LRP-2 regulates GLR-1 glutamate receptors and glutamatergic behavior in C. elegans

We identified the Low-Density Lipoprotein (LDL) Receptor Related Protein-2 (LRP-2) in a RNAi screen for genes that regulate glutamatergic behavior in C. elegans . lrp-2 loss-of-function mutants have defects in glutamatergic mechanosensory nose-touch behavior and suppress increased spontaneous reversals induced by GLR-1(A/T), a constitutively-active form of the AMPA-type glutamate receptor GLR-1. Total and surface levels of GLR-1 are increased throughout the ventral nerve cord of lrp-2 mutants suggesting that LRP-2 promotes glutamatergic signaling by regulating some aspect of GLR-1 trafficking, localization or function.


Description
We performed a RNAi screen for genes that regulate glutamatergic behavior as previously described (Luth et al., 2021). Mechanical stimulation of the nose of the worm with an eyelash activates a locomotion reversal behavior called the nose-touch response (Hart et al., 1995;Kaplan and Horvitz, 1993;Maricq et al., 1995). A pair of glutamatergic sensory neurons ASH, as well as other neurons, detect this nose-touch resulting in activation of GLR-1 AMPA-type glutamate receptors (AMPARs) on downstream interneurons such as AVA (Hart et al., 1995;Kaplan and Horvitz, 1993;Maricq et al., 1995;Mellem et al., 2002;Piggott et al., 2011). Expression of Channelrhodopsin specifically in ASH enables optogenetic activation of ASH, which results in glutamate-dependent calcium transients and depolarization of AVA interneurons and locomotion reversals (Ezcurra et al., 2011;Guo et al., 2009;Lindsay et al., 2011). We used this optogenetic ASH (optoASH) assay to photostimulate animals after RNAi knockdown of genes with cell adhesion molecule domains and monitored their reversal responses as described (Luth et al., 2021). We identified the Low-Density Lipoprotein (LDL) Receptor-Related Protein-2 (LRP-2) in this screen.
Our data suggest that lrp-2 is required for glutamatergic signaling in C. elegans. We found that lrp-2 mutants have defects in glutamatergic nose-touch behavior and suppress the increased spontaneous reversals induced by constitutively-active GLR-1(A/T). In mammals, LRP1 is highly expressed in the nervous system and localizes to the postsynaptic density (Nakajima et al., 2013). LRP1 can regulate AMPAR signaling and synaptic plasticity (Gan et al., 2014;Liu et al., 2010;Martin et al., 2008), however there are conflicting reports regarding its role in long-term potentiation (LTP) (May et al., 2004). Liu et al. (2010) reported that LRP1 is required for LTP in the hippocampus based on the conditional knockout of LRP1 in excitatory forebrain neurons in mice (Liu et al., 2010). In contrast, May et al. (2004) found no change in hippocampal LTP in a conditional knockout of LRP1 using synapsin-Cre mice (May et al., 2004). Another study found that LRP1 mediates the enhancement of LTP by Tissue-type Plasminogen Activator (tPA) (Zhuo et al., 2000). In this case the effect of LRP1 is likely mediated by direct interaction with the scaffold PSD95 and NMDA receptors (NMDARs) and regulation of NMDAR-mediated calcium signaling (Bacskai et al., 2000;Martin et al., 2008;May et al., 2004). LRP1 also interacts with AMPARs and controls their trafficking, however the role of LRP1 in regulating AMPAR trafficking is also controversial (Gan et al., 2014;Nakajima et al., 2013). Gan et al. (2014) showed that RNAi knockdown of LRP1 in cortical neurons leads to decreased total and surface levels of the AMPAR subunit GluA1 via increased internalization and degradation (Gan et al., 2014). In contrast, Nakajima et al. (2013) found that there was no change in surface or total levels of GluA1 under basal conditions in cortical neurons cultured from LRP1 knock-out mice. However, NMDA-induced internalization of GluA1 was dependent on LRP1 (Nakajima et al., 2013), suggesting that LRP1 is required for the endocytosis of AMPARs in response to NMDA treatment.
We found that total and surface levels of GLR-1 increase in vivo in lrp-2 mutants throughout the VNC in C. elegans. Given that LRP-2 promotes the internalization of a large number of proteins, our data might be consistent with a role for LRP-2 in promoting GLR-1 endocytosis. However, the fact that lrp-2 mutants also have defects in GLR-1-dependent behaviors suggests that the pool of GLR-1 that accumulates at the neuronal surface in lrp-2 mutants is not functional. Mammalian LRP1 can promote calcium signaling via GluA1 homomers (Gan et al., 2014) and NMDARs in cultured rodent neurons (Martin et al., 2008). Given that GLR-1 is permeable to calcium (Zheng et al., 1999) and LRP1 interacts with mammalian AMPARs (Gan et al., 2014), it is possible that LRP-2 may directly promote GLR-1 function in C. elegans. The increase in total GLR-1 observed in lrp-2 mutants could be due to a compensatory upregulation of GLR-1 expression, as we have previously shown happens in response to decreased glutamatergic signaling (Moss et al., 2016). We speculate that LRP-2 acts via more than one mechanism to regulate GLR-1 function, localization and/or trafficking.

RNAi screen
lrp-2 was found in a RNAi screen described in (Luth et al., 2021). Briefly, genes with cell adhesion molecule domains were knocked down using feeding RNAi in a strain enhanced for neuronal RNAi (FJ1300) and were screened in triplicate for defective backward movement in response to optogenetic activation of ASH sensory neurons as previously described (Luth et al., 2021).

Nose-touch assay
The nose touch assay was carried out on NGM plates that were spotted with OP50 (diluted 1:10 with LB broth) the day before the experiment and dried overnight. The experimenter was blinded to all genotypes. Individual animals were picked to the assay plate, then an eyelash attached to the end of a wooden stick was placed in the path of the worm. A trial was counted if the nose of the worm collided perpendicularly with the eyelash. A trial was scored as a reversal if the worm immediately initiated a backward movement a distance greater than the length of the nose to the posterior pharyngeal bulb. Ten trials were completed for each worm assayed.

Thrashing
Thrashing was performed as described previously (Luth et al., 2021). The experimenter was blinded to all genotypes. Briefly, individual worms were picked to a 5 µL drop of M9 Buffer on a glass coverslip and allowed to acclimate for ~1 minute. Following the acclimation period, a timer was set for 1 minute and the number of thrashes was counted using a handheld tally device. A thrash was defined as the worm moving through a C shape curled ventrally to dorsally and back.

Spontaneous reversal assay
NGM Assay plates were poured the day before the experiment and dried overnight. The experimenter was blinded to all genotypes. Individual animals were picked with halocarbon oil to the unspotted assay plate and were allowed to acclimate for 2 minutes. The number of spontaneous reversals in a 5-minute period were counted and recorded. Reversals were defined as backward movement greater than the distance from the nose to the posterior bulb of the pharynx.

Epifluorescence imaging and quantification
Imaging of GLR-1::GFP in nuIs24 was carried out on a Carl Zeiss Axiovert M1 microscope with a 100x Plan Apochromat objective (1.4 numerical aperture) as previously described (Luth et al., 2021). An 11 plane Z-stack with 1 µm total thickness captured the posterior VNC. A maximum intensity projection of the Z-stack was used to generate a linescan using Metamorph software (v7.1, Molecular Devices). Linescans were analyzed using custom-written software (IgorPro v6, Wavemetrics) (Burbea 2002, Kowalski 2011. Puncta were identified using a minimum peak width of 0.3 µm and a minimum peak threshold 4 standard deviations above the VNC background fluorescence. Puncta fluorescence intensities were normalized to the mean intensity of standardized fluorescent beads for each corresponding day of imaging. Puncta width was defined as the average of the widths of each punctum at half maximal peak intensity.

Confocal imaging and quantification
The VNC of worms expressing SEP::mCherry::GLR-1 (akIs201) was imaged using a Zeiss LSM800 confocal microscope with a 63X Plan Apochromat objective (numerical aperture 1.4). Identical acquisition settings were used for all images: 101.51 µm X 58.68 µm image size; z-stack: 18 slices (6.63 µm total thickness), 488 nm laser: 0.4% with 776V gain, 561 nm laser: 0.3% with 867V gain. Quantification of mCherry and SEP fluorescence was performed using a custom MATLAB script. The script was fed an average background fluorescence for the mCherry and SEP channels set from representative data sets, respectively, from wild type animals to threshold all images. The script facilitates drawing a ROI around the VNC that is applied to both the mCherry and SEP channels and used to measure total fluorescence within the ROI. For each imaging session, experimental genotypes were normalized to wild type so imaging sessions could be aggregated. Representative images were processed equivalently in ImageJ for display.